ObjectiveTo explore the light sensitivity and kinetic of the new optogenetics tools Channelrhodopsin-XXM2.0 (XXM2.0) and Channelrhodopsin-PsCatCh2.0 (PsCatCh2.0), and analyze whether they could be used to restore the visual function by optogenetics.MethodsMolecular biology techniques were used to link the gene fragments of XXM2.0 and PsCatCh2.0 to the vector pCIG(c)-msFoxn3 containing ampicillin resistant screening gene and reporter gene to form new plasmid pCIG(c)-msFoxn3-XXM2.0 and pCIG(c)-msFoxn3-PsCatCh2.0. The constructed plasmids were transfected into HEK 293T cells, and light responses were recorded in the whole cell mode with the HEKA patch clamp system. The photocurrent was recorded under three light intensity included 2.7×1016, 4.7×1015, and 6.4×1014 photons/(cm2·s). And then, XXM2.0 and PsCatCh2.0 were stimulated with 2.7×1016 photons/(cm2·s) and fully recovered. The opening and closing time constants were analyzed with Clampfit 10.6 software. At the same light intensity, photocurrents of XXM2.0 and PsCatCh2.0 were recorded by the light pulse stimulating of 2-32 Hz. The current attenuation was analyzed at long intervals of 4000 ms and short intervals of 200 ms after repeated stimulation. Comparisons between groups were performed by independent samples t test.ResultsRestriction endonuclease sites of EcoRⅠ and EcoRⅤ were successfully introduced at XXM2.0 and PsCatCh2.0 sequences. When the digestion was completed, they were ligated by T4 DNA ligase to construct new plasmids pCIG(c)-msFoxn3-XXM2.0 and pCIG (c)-msFoxn3-PsCatCh2.0, and then transfected on HEK 293T cells. The light intensity dependence was showed in XXM2.0 and PsCatCh2.0. The greater light intensity was accompanied by the greater photocurrent. Under the light intensity 6.4×1014 photons/(cm2·s) below the retinal safety threshold, large photocurrent was still generated in XXM2.0 and PsCatCh2.0 with 92.8±142.0 and 13.9±5.6 pA (t=1.24, 1.24; P=0.28, 0.29). The opening time constants of XXM2.0 and PsCatCh2.0 were 23.9±6.7 and 2.4±0.8 ms, and the closing time constants were 5803.0±568.2 and 219.9±25.6 ms. Compared with PsCatCh2.0, the opening and closing time constant of XXM2.0 were both larger than PsCatCh2.0. The differences were statistically significant (t=7.10, 31.60; P=0.00, 0.00). In terms of response frequency, XXM2.0 and PsCatCh2.0 could follow to 32 Hz high-frequency pulsed light stimulation, and all could respond to repeated light stimulation at a long (4000 ms) and a short time (200 ms) interval with the small current decay rate.ConclusionXXM2.0 and PsCatCh2.0 could be activated under light intensity with safety for the retina, and could respond to high frequency (at least 32 Hz) pulsed light stimuli with low current attenuation, which could meet the characteristics of opsins required to restore the visual function by optogenetics.
ObjectiveTo explore the light response, retinal inflammation and apoptosis of the retinal ganglion cells (RGCs) 1 year after the new type of channelrhodopsin PsCatCh2.0 was transfected into the retina of rd1 mice. MethodsTwenty-four male rd1 mice were randomly divided into rd1 experimental group and rd1 control group, 12 mice in each group. 1.5 μl of recombinant adeno-associated virus (rAAV)2/2-cytomegalovirus (CMV)-PsCatCh2.0-enhanced green fluorescent protein (EGFP) was injected into the vitreous cavity 1 mm below the corneoscleral limbus of mice in the rd1 experimental group, and the same dose of recombinant virus was injected 2 weeks later at temporal side 1 mm below the corneoscleral limbus. One year after virus injection, the light response of RGCs expressing PsCatCh2.0 was recorded by patch clamp technique; the expression of PsCatCh2.0 in the retina was evaluated by immunofluorescence staining; the transfection efficiency of recombinant virus was evaluated by the transfection efficiency of virus and the number of RGCs. Hematoxylin-eosin staining was performed to measure the inner retinal thickness. Western blotting was used to detect the protein expression of nuclear factor (NF)-κB p65 in retina; real-time quantitative polymerase chain reaction was used to detect the relative expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and Bax mRNA. Terminal deoxynucleotidyl transferase kit was used to observe the apoptosis of retinal cells in each group of mice. ResultsOne year after the intravitreal injection of recombinant virus, PsCatCh2.0-expressing RGCs can still generate 30 pA photocurrent. The virus PsCatCh2.0-EGFP was mainly transfected into RGCs, and partly transfected into amacrine cells, almost no transfection was seen in bipolar and horizontal cells. There were no significant differences in the number of RGCs and thickness of the inner retina between the rd1 experimental group and the rd1 control group (F=14.35, 0.05; P＞0.05), while the rd1 experimental group NF-κB p65 protein expression, TNF-α and IL-6 mRNA quantification were significantly lower than those of rd1 control group (F=4.61, 5.91, 5.78; P＜0.05). The number of red fluorescent apoptotic cells in the retina of mice in the rd1 experimental group was less than that in the rd1 control group, and the Bax mRNA expression was lower than that in the rd1 control group, and the difference was statistically significant (F=7.52, P＜0.01). ConclusionOne year after intravitreal injection of recombinant virus, the PsCatCh2.0 expressing RGCs can still generate photocurrent. Long term transfection and expression of PsCatCh2.0 has no obvious cytotoxic effect on RGCs, nor it increases the inflammatory effect of the retina of rd1 mice with retinal degeneration.
Objective To systematically evaluate the influence of PICC (peripherally inserted central catheter) ultrasound guidance on success rates and incidences of complications of elbow catheterization. Methods The Cochrane Library (Issue 3, 2012), Library of JBI evidence-based nursing center, MEDLINE, EMbase, CBM, VIP, CNKI and WanFang Data were searched for relevant studies to influence on success rates of puncture and catheterization and complications from upper arm placement of PICC ultrasound guidance as well as conventional PICC placement from inception to March, 2012. The data were extracted according to the inclusion and exclusion criteria by two reviewers, the quality of included studies was also evaluated. Meta-analysis was conducted using RevMan 5.1, or only descriptive analysis was conducted instead. Results Eleven randomized controlled trials, five clinical controlled trials and four cohort studies were included, involving 4 052 patients. The results of meta-analysis showed that, upper arm placement of PICC ultrasound guidance combined with modified Seldinger technique was associated with significantly higher one-time success rates of puncture and catheterization than conventional PICC placement below the elbow, with significant differences (OR=4.71, 95%CI 1.97 to 11.28, P=0.000 5; OR=8.63, 95%CI 1.92 to 38.82, P=0.005). Conventional elbow catheterization had a significantly higher incidence of phlebitis than catheterization on the elbow that under the guidance of PICC ultrasound and modified Seldinger technique (OR=0.13, 95%CI 0.08 to 0.21, Plt;0.000 01). Conclusion Elbow catheterization under the guidance of PICC ultrasound combined with modified Seldinger technique has an obvious advantage in improving the success rates of puncture and catheterization and reducing the incidence of complications, compared with conventional elbow catheterization.
ObjectiveTo explore etiopathogenesis of slow transit constipation(STC). MethodsThe model of rat with “cathartic colon” was established, and the changes of colonic electromyography of the rat was examined. ResultsThe frequency and amplitude of slow wave in vivo of rats with cathartic colon was decreased markedly. Postmeal spike potential and duration was decreased, showing that gastrocolonic reflex of rats colon was decreased.ConclusionLong term abuse of stimulant laxuatives could damage enteric nervous system and accelerate the pathological changes of STC.
Objective To investigate the expression of cerb B2 and CathepsinD in gastric carcinoma and its correlation with the biological behavior of gastric carcinoma (GC). MethodsThe expression was studied by immunohistochemical technique. The expression of cerb B2 and CathepsinD were analyzed with their relation to histologic types, depth of invasion, growth pattern, lymph node metastasis and prognosis of gastric carcinoma. ResultsThirtynine of the 102 gastric carcinoma specimens (38.24%) were positive for cerb B2 and correlated with depth of invasion (P＜0.05) and lymph node metastasis (P＜0.05); eightythree of the 102 gastric carcinoma specimens (81.37%) were positive for CathepsinD and correlated with depth of invasion (P＜0.05), growth pattern (P＜0.05), lymph node metastasis (P＜0.05) and blood vessels cancer embolus (P＜0.05). Prognosis of patients with gastric carcinoma with positive expression of cerb B2 or CathepsinD was poor. The 5year survival rate was significantly lower in gastric carcinoma patients with positive expression of cerb B2 or CathepsinD. Conclusion cerb B2 and CathepsinD are highly related to growth, invasion, metastasis and prognosis of gastric carcinoma.
Objective To discuss the molecular structures and functions of βCatenin and APC (Adenomatous polyposis coli) genes, as well as the roles they played during the carcinogenesis and progression of tumors. MethodsAdvances in molecular biology and oncology of βCatenin and APC genes in recent years has been reviewed. ResultsHigh expression of βCatenin and APC genes was an early event during the carcinogenesis due to the abnormal regulation of βCatenin and APC genes. But on the contrary, the low expression of these two genes was probably associated with the progression of tumors. Moreover, βCatenin and APC genes could also regulate the expression of p53,cmyc genes and cyclin D1. Conclusion βCatenin and APC genes may play a key role during the carcinogenesis and progression of tumors and modulate other oncogen/suppressor gene and factors.
【Abstract】Objective To investigate the diagnostic and therapeutic value of selective angiography and embolization treatment in massive biliary hemorrhage in emergency. Methods Eleven cases of massive biliary bleeding were selectively angiographied and embolized with thread segment and gelatin sponge grain by Seldinger technique in emergency. Results Massive biliary bleeding in this series included bleeding after biliary surgery (7), post-laparoscopic cholecystectomy bleeding (1), bleeding from traumatic injury to liver (1), and rupture of huge hepatic cancer (2). There were 9 cases with abnormal angiographic findings including contrast medium leakage (9), tumor vessels and tumor tissue staining (2) and signs of pseudo-aneurysm formation (4). All the bleedings were stopped after emergency intervention but 2 cases with recurrent massive bleeding after 48 hours. Conclusion Emergency trans-catheter embolization treatment is an effective and safe method for biliary massive hemorrhage. It is also important to keep the biliary tract unobstructed.
To evaluatae the role of cathepsin B in the pathogenesis of acute pancreatitis. The experimental dogs were divided into three groups based on the severity. An acute edematous pancreatitis group (n＝11), an acute hemorrhagic necrotizing pancreatitis group (n＝12), and a control group (n＝7). Distribution of cathepsin B in pancreatic aciner cell was studied. Results: there was a redistribution of cathepsin B from the lysosomal fraction to the zymogen fraction. The results indicated that cathepsin B play a important role in the pathoagenesis of acute pancreatitis.
Objective To investigate the species distribution and antibiotic resistance of pathogens fromcatheter-related bloodstream infections ( CRBSI) in intensive care unit( ICU) , to provide evidence for the guidance of clinical rational administration.Methods A retrospective analysis was performed to review the microbiological and susceptibility test data of all CRBSI patients in ICU from January 2009 to December 2011. The patterns of antibiotic resistance among the top seven bacteria were compared. Results 67 cases of CRBSI were detected with 81 strains, including 40 Gram-positive ( G+ ) bacteria( 49.4% ) , 38 Gram-negative( G- ) bacteria ( 46.9% ) , and 3 fungi ( 3.7% ) . The main pathogens causing CRBSI were coagulase negative Staphylococci ( 27 strains, 33.3%) , Acinetobacter baumannii ( 12 strains, 14.8% ) , Klebsiella pneumoniae( 9 strains, 11. 1% ) , Staphylococcus aureus ( 8 strains, 9. 9% ) , Pseudomonas aeruginosa ( 7 strains, 8. 6% ) , Escherichia coli ( 6 strains, 7.4% ) , suggesting that Staphylococcus epidermidis was predominant pathogenic G+ bacteria, and Acinetobacter baumannii was predominant G- bacteria. The antibiotic resistance tests demonstrated that isolated G- bacillus was highly sensitive to carbopenem, while vancomycin-resistant G+ bacteria were not found. Conclusions Within the latest 3 years, the predominant pathogens of CRBSI in ICU are Staphylococcus epidermidis and Acinetobacter baumannii. Acinetobacter baumannii exhibited high drug resistance to all antibiotics.
Objective To explore the risk factors of catheter-associated bloodstream infections ( CRBSI) in intensive care unit ( ICU) of primary hospital. Methods A total of 623 patients with central venous catheters were recruited in the study. 60 of themsuffered fromCRBSI served as an observation group and other 563 cases without CRBSI served as control. Univariate analysis was used to scan possible risk factors. Then logistic regression analysis was used to exclude the confounding factors. Results The overall incidence rate of CRBSI was 9. 63% ( 60 /623) . There were significant differences in APACHE score, type of catheter, location of catheter, duration of central venous catheter, intravenous nutrition, use of steroid, times of intubation, urgent intubations, nutritional status, diabetes, and MODS between the two groups. Logistic regression analysis revealed that higher APACHE score, double-lumen catheter, femoral vein catheter, catheter indwelling more than two weeks, intravenous nutrition, intubation more than 2 times, and emergency intubation were risk factors of CRBSI. Conclusions Higher APACHE score, double-lumen catheter, femoral vein catheter, catheter indwelling more than two weeks, intravenous nutrition, intubation more than 2 times, and emergency intubation were major risk factors of CRBSI in ICU of primary hospital.